1. Technical Field
The various embodiments of the present disclosure relate generally to single molecule sensitive probes for detecting RNA. More specifically, the various embodiments of the present disclosure are directed to multivalent fluorescent probes for detecting a single molecule of RNA in a living or fixed cell or tissue.
2. Description of Related Art
In recent years, the imaging of RNAs in live cells has garnered growing attention with a variety of interesting methods developed for this purpose. This research has been driven by the increased appreciation for the role of post-transcriptional regulation on the function of RNA molecules, the role of genomic RNA in viral assembly, and the cellular stress response in pathogenesis.
Various technologies and methodologies have been developed to study intracellular RNA biology in live cells including: plasmid-based systems, where the fluorescent tag and/or the RNA to be tagged are expressed within the cell, or systems based on exogenous RNA-targeting fluorescent probes, which when introduced into the cell, bind to their target via Watson-Crick base pairing. Currently, expressing both the RNA and a fluorescent tag using a plasmid-based system is the state-of-the-art in this field.
Three strategies using plasmid expressed probes have been demonstrated in mammalian cells: (1) a GFP-MS2 fusion protein probe, which binds to multiple binding sites encoded in an expressed target RNA; (2) GFP-RNA binding peptide fusion probes, which bind to a 15 nucleotide RNA hairpin encoded in the expressed target RNA; and (3) probes composed of Pumilio homology domains (PUM-HD) fused to sections of split EGFP, which target two closely spaced 8 nucleotide endogenous sequences. These systems have been used to study cytoplasmic, nuclear mRNA and mitochondrial RNA. A fourth technique, which is an extension of the first three, introduces an exogenous probe, a microinjection-delivered molecular beacon that targets multiple binding sites in an expressed target mRNA.
Employing plasmid-derived probes and RNA give these methods tremendous flexibility but they have limitations. First, they can only be used in cell types that allow for efficient transfection. Second, plasmid-derived mRNA often lack introns, both the correct number and position and the exact 3′-untranslated region (UTR) sequence, which can strongly influence mRNA translational efficiency, decay, and stability. In the case of viral RNA, additions to viral genomes affect replication efficiency, assembly, and viral egress. In addition, plasmid-derived RNAs are often overexpressed, changing the fundamental stoichiometry underlying the RNAs expression. Therefore, it is advantageous to target endogenous RNA in order to improve RNA biology studies. Of the techniques mentioned above, only the PUM-HD fusions and the molecular beacon approach have the ability to study endogenous or non-engineered RNAs, but neither have achieved single molecule sensitivity with endogenous targets.
When imaging endogenous RNA, the sensitivity of the probe is extremely important. First, mRNA is not highly abundant within cells, and second, RNA function is governed by the set of highly abundant proteins that interact with it. The difference in their concentrations makes studying these interactions via imaging very difficult. Therefore, RNA probes must be sensitive enough to detect a small number of RNAs within a sea of proteins. In order to achieve single molecule sensitivity, the above techniques require the binding of many probes; for example, binding sites for up to 50 MS2-GFP molecules or 96 molecular beacon molecules are necessary. Given both the additions to the RNA and the size of the probes, large molecular weight additions are required to achieve single molecule sensitivity. These additions could have effects on both RNA localization and dynamics. Further, for probe-based methods, delivery into the correct cellular compartment is critical in order for the probes to bind rapidly to their target. Delivery methods that result in accumulation of probe in the nucleus of cells or utilize endocytic vesicles can lead to nonspecific signal and degradation of the probe.
In order to make an advance in the area of RNA imaging, new techniques must be capable of imaging endogenous RNA, have single molecule sensitivity, allow for the imaging of multiple RNAs, and minimize the number and molecular weight of probes binding to the RNA. It is to the provision of such RNA imaging techniques that the various embodiments of the present invention are directed.